IS 12572 ( Part 7 ) : 1988

Indian Standard

( Reaffirmed 2006 )

GUIDE FOR EVALUATION OF MEDICAL DEVICES FOR BIOLOGICAL HAZARDS
PART 7 METHODS OF TEST FOR SENSITIZATION : ASSESSMENT OF POTENTIAL OF MEDICAL DEVICES TO PRODUCE DELAYED CONTACT DERMATITIS

UDC 615'4 ; 614'8 : 616-022 : 616-5-002

DESCRIPTORS : MEDICAL DEVICES. BIOLOGICAL HAZARDS SENSITIZATION.

POTENTIAL OF MEDICAL DEVICES. CONTACT DERMATITIS,

@ BIS 1989

BUREAU

OF

INDIAN

STANDARDS

MANAK BHAVAN, 9 BAHADUR SHAH ZAFAR MARG NEW DELHI 110002 September 1989
Price Group 3

Medical Glass Instruments and Appliances Sectional Committee, CPDC 12

FOREWORD This Indian Standard ( Part 7 ) was adopted by the Bureau of Indian Standards on 14 September 1988, after the draft finalized by the Medical Glass Instruments and Appliances Sectional Committee had been approved by the Consumer Products and Medical Instruments Division Council. This part ( Part 7 ) of the standard is one of a series of methods of test for assessing the biological hazards of medical devices that directly contact body tissues or substances that are to be introduced into the body by means other than oral. This standard describes methods of test for sensitization; assessment of the potential of medical devices to produce delayed contact dermatitis, and should be read in conjunction with Part 2 of this standard. The testing of the ingredients of the material from which a device is made, has been included because sensitization tests using ingredients may be more indicative of potential clinical response than testing only the final product itself. This standard is proposed to be published in 12 parts. Other parts are as follows: Part 1 Terminology Part 2 Selection of biological methods of test Part 3 Method of testing by tissue implantation Part 4 Method of test for systeniic toxicity; Assessment of acute toxicity of extracts from medical devices Part 5 Method of test for intracutaneous reactivity of extracts from medical devices Part 6 Method of test for systemic toxicity; Assessment of pyrogenicity in rabbits of extracts from medical devices Part 8 Method of test for skin irritation of extracts from medical devices Part 9 Method of test for skin irritation of solid medical devices Part 10 Method of test for dental materials Part 11 Method of test for eye irritation Part 12 Method of test for toxicity to cells in culture of extracts from medical devices Compliance with this standard does not in itself confer immunity from legal obligations. In the preparation of this standard, assistance has been derived from BS 5736 : Part 6 : 1983 `Evaluation of medical devices for biological hazards: Part 6 Methods of test for sensitization; assessment of the potential of medical devices to produce delayed contact dermatitis', issued by the British Standards Institution, UK.

IS 12572 ( Part 7 ) : 1988

Indian Standard
GUIDE FOR EVALUATION OF MEDICAL DEVICES FOR BIOLOGICAL HAZARDS
PART 7 METHODS OF TEST FOR SENSITIZATION : ASSESSMENT OF POTENTIAL OF MEDICAL DEVICES TO PRODUCE DELAYED CONTACT DERMATITIS 1 SCOPE 1.1 This standard ( Part 7 ) prescribes methods of 2.4 Erythema A tissue reaction characterized by redness at the

test designed to provide information on the potential to induce delayed contact dermatitis ( Type IV sensitization ) attributable to endogenous or extraneous substances present in or on a medical device. The methods specify grading of the skins reaction produced in guinea pigs following administration of prepared ingredients, and/or extracts of the device prepared using both a polar and a non-polar solvent. 1.2 Two test methods are prescribed: a) Method I uses a combined intradermal injection and topical application, b) Method 2 uses topical application only. Method 1 is the preferred method. Method 2 is to be used as an alternative to Method 1 if intradermal injection is not possible. 1.3 These methods of test are recommended for the initial assessment of all the categories of devices given in Part 2 of this standard. 2 DEFINITIONS For the purpose of this standard, the following definitions shall apply. 2.1 Challenge Treatment of the test animal after induction so as to demonstrate the presence or absence of sensitization. 2.2 Delayed Contact Dermatitis A cutaneous manifestation of Type IV sensitivity elicited by contact between an allergenic substance in the skin of : ensitized animals. 2.3 Device, Medical Device An item used in medic:ll treatment, diagnosis or ContmcePtion, not intended to have a pharmacological action on the body.
1

injection or application site, 2.5 Escbar A tissue reaction characterized by scab formation at the injection or application site. 2.6 Final Product A medical device in its ready-for-use state. 2.7 Induction Treatment of the test animals so as to create the opportunity for development of sensitization. 2.8 Ingredient A substance used in the formulation of the material from which the final product has been made. 2.9 Intradermal Injection An injection into the dermis of the skin. 2.10 Oedema A tissue reaction characterized by swelling at the injection or application site.
2.11 Prepared Ingredient

A solution or suspension of an ingredient or appropriate mixture of ingredients of the material from which the device under test has been made. 2.12 Test Material The final product or a sample of the final product that has to be tested. 2.13 Type IV Sensitization A cell-mcdinted immune response to allerg&ic substances involving T-lymphocytes and characterized by a delayed skin reaction It is distinguished from Type I to III reactions which are ilntibody-mediated.

IS 12572 ( Part 7 ) : 1988 3 ANIMALS AND HUSBANDRY 3.1 A minimum of thirty-six ( Method 1 ) or a minimum of thirty-four ( Method 2 ) albino given pigs of either sex from a single outbred strain, weighing 350 to 550 g at the start of the test, shall be used. 3.2 It may be necessary to use more than six animals ( Method 1 ) or four animals ( Method 2 ) for the preliminary tests. Thirty animals are required for each extract of the test material or prepared ingredient used in the induction phase of Method 1 and Method 2 ( see 5.3.2 and 5.1.2 ). 3.3 The animals shall have free access to food and water, and may be group caged. 4 PREPARATION OF EXTRACTS AND INGREDIENTS 4.1 Test Materials 4.1.1 Test material from pre-sterilized ( refers to devices obtained sterilized, from the manufacturer or supplier ) devices shall be handled aseptically throughout the extraction and test procedure. 4.1.2 Test material from devices that are normally supplied non-sterilized but are sterilized before use, shall be sterilized by the procedure normally used before the extraction and handled aseptically throughout the extraction and test procedure. 4.1.3 Test material from devices not required to be sterile in use shall be subjected to the extraction procedure and the extract obtained shall then be sterilized before administration. 4.2 Extractants The following are required for the preparation of extracts and for use as controls. 4.2.1 Polar Solvent Sterile saline solution containing 9 g/l sodium chloride. 4.2.2 Non-Polar Solvent Liquid paraffin IP/BP or other suitable solvent. 4.3 Principles of Extraction 4.3.1 The test material shall be representative of the final product, or of that component of the tinal product which is to be tested. 4.3.1.1 To achieve this, the test material may consist either of small pieces of the final product, or of one or more intact devices, For multicomponents devices, for example, dialysis units or 2 memberance oxygenators, it may be appropriate to test the individual components of the device separately. 4.3.1.2 The quantity of any endogenous or extraneous substances extracted is related to the surface area of the sample ( interfacial area ) with which extractant is brought into contact. 4.3.1.3 The concentration of any endogenous or extraneous substances in the extract and hence the dose delivered to the test animal, depends on both the interfacial area and the final extract volume, 4.3.1.4 The interfacial area per unit volume of the extract has to be taken into consideration when the dose given to the test animal is related to the likely exposure of the device to man. 4.3.2 To render the test as sensitive as possible, the ratio between interfacial area and the final extract volume shall be as high as is practicable. 4.3.2.1 The value of this test is enhanced if induction and challenge phases are performed using the maximum permissible concentrations ( see 5.3.1 and 5.4.1 ). 4.3.3 The technique to be used in exposing the , test material to the extractant shall be selected according to the requirements of 4.3.1 and 4.3.2, and may consist of total immersion or flowthrough methods, or a successive extraction of more than one specimen of the final product. 4.3.3.1 Fragmentation of the test material may be used to increase the interfacial area. 4.3.4 Extraction conditions shall simulate, as closely as possible, the conditions under which the device will normally be used. 4.3.5 During the extraction, 27 3: 3°C shall be representative of room temperature and 37 & 2°C that of the body temperature. 4.3.6 The period of contact between extractant and test material shall be 72 h. 4.4 Procedure 4.4.1 Using both the polar and the non-polar solvent as extractants, prepare in accordance with 4.1 and 4.3, sufficient volumes of extracts of the test material for administration as specified in 5. 4.4.2 In conditions identical with those used in 4.4.1, prepare controls using both polar and the non-polar solvent, in the absence of test material.

IS 12572 ( Part 7 ) : 1988
4.4.3 Sterilize extracts obtained from unsterilized

devices ( see 4.1.3 ). 4.4.4 Record in the test report, the precise conditions of extraction and, if applicable, of sterilization. 4.5 Preparation of Ingredients 4.5.1 Using a non-irritant vehicle, dissolve or suspend the ingredient at a suitable initial concentration ( see 5.3.1 and 5.4.1 ). 4.5.2 Suitable mixture of ingredients may be used. The preferred vehicles are the extractants specified in 4.3. It is only necessary to test with one vehicle. 5 TEST PROCEDURE 5.0 The tests described in this clause may be carried out on extracts of the test material ( see 4 ) and/or prepared ingredients ( see 4.5 ), as appropriate. If prepared ingredients are not used, the tests should be carried out on both the polar and the non-polar solvent extracts of the test material. 5.1 Preparation of Animals Clip all treatment sites and shave those for topical application, at least 24 h before treatment. 5.2 Administration of Extracts and Prepared Ingredients For all intradermal injections. inject into the clipped interscapuiar region of the animal, using 0.1 ml per injection site. 5.2.1 For all topical applications, saturate a patch of filter paper or surgical gauze of approximate dimensions 20 mm x 20 mm or 20 mm x 40 mm with the extract of the test material or the prepared ingredient and apply the patch to the clipped and shaved skin surface under a retaining dressing. 5.3 Method 1 5.3.1 Preliminary Test 5.3.1.1 This test determines the concentration of extracts of the test material and/or of' prepared ingredients to be used in the main test described in 5.3.2. 5.3.1.2 Pre-treat six animals by making a pair of intradermal injection of Freund's complete adjuvant in IP or BP grade water for injection (50 : 50 ) into the interscapular region of each animal.

5.3.1.3 Leave the pre-treated animals for a period of between 1 week and 3 weeks before continuing the test. 5.3.1.4 If necessary ( see 5.3.1.7 ), prepare a suitable dilution of the extracts of the test material in the corresponding extractants, or of the prepared ingredients in the corresponding vehicles. 5.3.1.5 Inject the extracts or the prepared ingredients or dilutions of these, intradermally into two of the pre-treated animals, 5.3.1.6 Note the appearance of the injection sites during following 48 h. Select: a) the highest concentration that causes no more than a local moderate to severe erythema ( numerical rating not higher than 3, see Table 1). Topically apply, using 20 mm x 20 mm patches, the extracts or the prepared ingredients or diluters of these, to the flanks of the four remaining pre-treated animals. Remove the dressings and patches after 24 h and note the appearance of the application sites. Select : b) the highest concentration that causes no more than very slight erythema and/or oedema ( numerical rating not higher than 1, see Table 1 ). Table 1 Rating System for Skin Reactions [ Clauses 5.3.1.6 (a J, 5.3.1.6 (b), 5.3.1.6 (c), 5.3.3.1, 5.4.1.4 ( a ), 5.4.1.4 ( b ), and 5.4.4 ]
Reaction Erythema and Eschar Formation No erythema Very slight erythema ( barely Numerical Rating 0

perceptible ) Well defined erythema Moderate to severe erythema Severe erythema ( beet redness ) to slight eschar formation

1
2

3 4

Oedema Formation No ocdema 0 Very slight oedema (barely 1 perceptible ) WCII defined oedema ( edges 2 of arca well defined by definite raising ) Moderate oedema ( raised 3 approximately 1 mm ) Severe oedema (raised more 4 than 1 mm and extending beyond exposure area ) ~<)~~-Other adverse changes :tt t h e kin site should be rccordcd anti reporled.

3

IS 12572 ( Part 7 ) : 1988 c) the highest concentration ( see 5.3.1.8 )

that is expected to cause neither erythema nor oedema ( numerical rating 0, see Table 1 ), when topically applied to the control animals in the challenge phase of the main test. 5.3.1.7 The limitations of the methods of extraction are such that the undiluted extracts of the test material will not normally cause any reaction

NOTE-Ideally the final concentractlon of the extract or of the prepared ingredient in ( 3 ) should be same as that in 5.3.1.6 ( b ), but this will not normally be possible for extracts ( see 5.3.1.7 ).

b) One week after the intradermal injections,

following intradermal injection or topical application. In such circumstances, the undiluted extracts should be used in the main test.
5.3.1.8 Suitable allowances should be made for variation in the response of the individual animals when predicting the concentration in 5.3.1.6 ( c ). 5.3.1.9 Any systemic effects resulting from

administer the extracts or the prepared ingredient by topical application, using 20 mm x 40 mm patches, to the interscapular region of each animal, so as to cover the intradermal injection sites. Use the concentration selected in 5.3.1.6 ( b ). Remove the dressings and patches after 48 h. the corresponding control extract or the vehicle alone.

4 Treat the control animals similarly using
5.3.2.3 Challenge phase
12 days after completion of the induction phase, challenge all the test and control animals with the prepared ingredient or the extract. Administer the prepared ingredient or the extract by topical application using 20 mm x 20 mm patches to one flank of each animal, using the concentration selected in 5.3.1.6 ( c ). Remove dressings and patches after 24 h.

administration at the concentration selected in 5.3.1.6 ( a ), ( b ) and ( c ) should not be such as to impair the health of the animal.

5.3.1.10 Additional animals may be used to clarify equivocal results. 5.3.2 Main Test 5.3.2.1 General Use a minimum of twenty animals as a test group and a minimum of ten animals as a control group. Except for the ommission of the extract or of the prepared ingredient during the induction phase, treat the control animals in the same in this manner as the test animals throughout the test. 5.3.2.2 Induction phase Use twenty test animals and ten control animals for each extract or each prepared ingredient used in this phase. a) Make a pair of intradermal injections of each of the following, into each animal, at the injection sites shown in Fig. 1:

a) If the induction phase was performed

using a prepared ingredient, the animals may be challenged both with each extract of the test material and with the prepared ingredient.

b) Additional challenging with the greater
dilution of the prepared ingredient or the extract than selected in 5.3.1.6 ( c ) may facilitate the interpretation of the reactions.

5.3.3 Observation of Animals 5.3.3.1 Note the appearance of challenge skin sites

1)

Freund's complete adjuvant emulsion in IPBP grade water for injection ( 50 : 50 ). at the concentration selected in 5.3.1.6 ( a ). Inject the control animals with the corresponding control extracts or vehicle alone.

of the test and control animals within 1 h after removal of the dressing and subsequently after 24, 48 and 72 h ( that is, 25,48,72 and 96 h after the initial challenge application ). 5.3.3.2 Describe and rate the skin reactions of erythema and oedema according to the rating system given in Table 1 for each challenge site and at each time interval observed. Record the results for the test report. 5.4 Method 2 5.4.1 Preliminary Test
5.4.1.1 This test determines the concentrations of the extracts of the test material and/or of the

2) The extract or the prepared ingredient

3) The extract or the prepared ingredient emulsified in Freund's complete adjuvant in IPBP grade water for injection ( 50 : 50 ). Inject the control animals with the corresponding control extract or vehicle, emulsified in adjuvant.

4

IS 12572 ( Part 7 ) : 1988
HEAD O-1 ml INTRAOERMAL INJECTIONS

-- -/-I

I1 :8'81 1 11

1

\

CLIPPED INTERSCAPULAR REGION

REAR

FIG. 1 L OCATION

OF

INTRADERMAL INJECTION S ITES

FOR

M ETHOD 1

prepared ingredients to be used in the main test described in 5.4.2. 5.4.1.2 If necessary ( see 5.4.1.5 ), prepare the suitable dilutions of the extracts of the test material in the corres ponding extractant, or of the prepared ingredient in the corresponding vehicle.
5.4.1.3 Topically apply, using 20 mmx20 mm

541.5 The limitations on the methods of extraction are such that the undiluted extracts of the test material will not normally cause any reaction
following topical application. In such instances, the undiluted extracts should be used in the main test. 5.4.1.6 Suitable allowance should be made for variation in the response of individual animals when predicting the concentration in 5.4.1.4 ( b ). 5.4.1.7 Any systemic effects resulting from administration at the concentration selected in 5.4.1.4 ( a ) and ( b ) should not be such as to impair the health of the animal. 5.4.1.8 Additional animals may be used to clarify equivocal results. 5.4.2 Main Test 5.4.2.1 General Use minimum twenty animals as a test group and minimum ten animals as 3 control group. 5.4.2.1.1 Except for the omission of the extract 01' the prepared ingredient during the ~r~clwt ion 5

patches, the extracts or the prepared ingredients or dilutions of these, to the flanks of four animals. 5.4.1.4 Remove the dressings and patches after 6 h and note the appearance of the application s~:s. Sz 1 ect: a) the highest concentration that causes no more than very slight erythema and/or oedema ( numerical rating not higher than 1, see Table 1 ); and b) the highest concentration ( .W 5.4.1.6 ) that is expected to cause neither erythema nor o&ma ( numerical rating 0, see Table 1 ), when topically app!icd to the control
animals in the chalicnge phase of the main test.

%S 12572 ( Fart 7 ) : 1988 phase, treat the control animals in the same manner as the test animals throughout the test. 5.4.2.2 Induction phase Use twenty test animals and ten control animals for each extract or each prepared ingredient used in this phase. a) Administer the extract or the prepared ingredient by topical application using 20 mm% 20 mm patches to the left shoulder region of each animal. Use the concentration selected in 5.4.1.4 ( a ). Remove the dressings and patches after 6 h. b) Repeat this procedure on alternate days 3 days each week for 3 weeks ( that is, total nine applications ). removal of the dressing and subsequently after 24, 48 and 72 h ( that is, 7, 30 and 78 h after initial challenge application ). 5.4.4 Describe and rate the skin reactions of erythema and oedema according the rating system given in Table 1 for each challenge site and at each time interval observed. Record the results for the test reports. 6 PRESENTATION OF RESULTS 6.1 The results shall be submitted in a test report that includes a complete record of the method of extraction and preparation, the procedures followed, and any relevant data necessary for the assessment of results as described in 7.

c) Treat the control animals similarly using 7 ASSESSMENT OF RESULTS the corresponding control extracts or 7.1 Assessment of the results should be carried vehicle alone. out by an experienced person ( preferably a 5.4.2.3 Challenge phase toxicologist ) who is aware of the conditions of of the final product and has appropriate Two weeks after the ninth induction application, ose chemical and biological data concerning it ( see challenge all test and control animals with the Part 2 of this standard ). prepared ingredient or the extract. Administer the prepared ingredient or extract by a single 7.2 Limitations of methods of extraction and topical application, using 20 mm x 20 mm patches, testing are such that tests on the final product to one flank of each animal, using the concentra- alone may not be useful in some cases. Tests tion selected in 5.4.1.4 ( b ). Remove the patches using prepared ingredients may supply more and dressing after 6 h. information and should be carried out, where a) If the induction phase was performed practicable. using a prepared ingredient, the animals may be challenged both with each extract 7.3 When ingredients are investigated, they of the test material and with the prepared should be prepared in suitable administration media. ingredient. b) Additional chahenging, with greater dilu- 7.4 The overall assessment of the test results tions of the prepared ingredient or of the shall be carried out,taking into consideration any extract of final product than selected difference observed for the incidence and severity in 5.4.1.4 ( b ) may facilitate the intcr- of dermal reactions between the test and the pretation of the reactions. control animals. If the assessing person ( or toxicologist ) considers the results to be either 5.4.3 Observation of Animals inconclusive or invalid, the test shall be repeated Note the appearance of the challenge skin sites using different animals, new extract-s/preparat-ions of test and control animals within 1 h after and, if necessary, different extra&ants/vehicles.

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